This invent ion relates to a multi-well cluster plate for growing cells or tissue cultures in vitro and more particularly comprises a new and improved cell culture plate and well structure that combines a device for supporting tissue cultures in a fluid medium with a structure that allows unfettered access to the portion of the well below the tissue culture.
Advantages of growing tissue in vitro by supporting a microporous membrane as a cell growth substrate in a culture medium have been known. By supporting a microporous membrane in a nutrient rich medium, a cell layer can be attached and grown on the membrane. This cell layer is able to receive nutrients through the microporous membrane from the medium below. The cell layer is separated from the medium by the membrane. A concentration gradient of nutrients develops and feeds the cells through this permeable membrane. This basolateral method of feeding more closely resembles in vivo conditions in which polarized structures such as epithelial cells functionally behave. Creating more natural cellular conditions is an advantage to scientists studying cell transport and other biological activities.
Cell culture inserts have been used to take advantage of the basolateral feeding technique. Cell culture inserts are comprised of a plastic material with a membrane on the bottom surface. The inserts fit into wells of a culture plate, such that the membrane is suspended within a corresponding well. The well itself contains culture medium into which the metr.-rane is immersed. Conventional cell culture inserts and devices are described in U.S. Pat. No. 4,871,674, U.S. Pat. No. 5,026,649, U.S. Pat. No. 5,215,920, U.S. Pat. No. 5,466,602, and U.S. Pat. No. 5,468,638. Many inserts also include an opening in the hanging support structure which enables access to the culture medium below the membrane. This access is crucial for maintaining appropriate levels of nutrients and waste products in the culture medium. Through these openings, culture medium can be removed and replaced without disturbing the membrane substrate.
U.S. Pat. No. 5,141,718 discloses an apparatus that combines a strip of insert wells with a plate containing tear shaped reservoirs. Each reservoir is comprised of a circular portion and a triangular portion. The strips are comprised of a plurality of tubular members attached to membranes. These strips are inserted into the plate such that each tubular member is immersed in a reservoir. The tubular member occupies substantially all of the circular portion of the reservoir. Access to the reservoir is accomplished through the triangular portion, without disturbing the membrane substrate. The problem with this insert strip plate apparatus is twofold. birst, the wells do not conform to the standard 96-well industry standard and thereby are not adaptable to much of the auxiliary equipment designed specifically for such plates. Second, the insert strips are comparatively unstable when measured against a plate that has its entire well structure permanently integrated into the plate itself.
The drug industry has for several years used inserts, such as shown in U.S. Pat. No. 5,026,649, for use in drug transport studies. Cells are grown to a confluent monolayer on a microporous membrane. Electrodes can be placed on either side of the membrane in order to test the confluency of the monolayer. Thereafter, a compound to be screened is added to the lower portion of the well, below the membrane. After a suitable incubation time, an assay of the solution on the other side, above the membrane, is performed. The presence and concentration of the compound on the other side of the membrane indicate- the compound's ability to transport through the particular cells comprising the monolayer. A need exists for a culture plate capable of performing these types of cell transport studies on an industrial scale for the screening of large numbers of drugs.
Industry standard microtest cluster plates are laid out with 96 wells, each having an approximate cross sectional area of 0.053 squre inches, in an 8.times.12 matrix (mutually perpendicular 8 and 12 well rows) with a spacing of 0.355 inches between the center lines of rows, both in the x and y directions. In addition, the height, length and width of the microtest 96-well plates are standardized. This standardization has resulted in the development of a large array of auxiliary equipment specifically developed for 96-well formats. The equipment includes devices that load and unload precise volumes of liquid in multiples of 8, 12, or 96 wells at a time. In addition, equipment is available to transmit light through individual wells and to read calorimetric changes or chemiluminescence in individual wells. Some of this equipment is automated and instrumented to record, analyze and manipulate the data recorded.
A problem associated with insert devices is that their size does not allow them to easily conform to the standard 96-well culture plate format. Insert devices suspend a membrane within a well. In order to suspend the membrane without it touching the walls of the well, the membrane itself must be substantially smaller in diameter than the diameter of the inside of the well. Further, access to the lower part of the well is accomplished by inserting a pipette or syringe to the side of the suspended membrane. Therefore, the suspended membrane must be not only small enough to prevent contact with the wall of the well, but also small enough to allow a pipette or syringe to fit between it and the well wall. In a 96 well plate, the diameter of the wells is relatively small (approximately 0.325 inches). Inserts that fit into these small wells have membranes that are considerably smaller in diameter than the wells. These small membranes are difficult to handle and perform studies upon.
The insert strips of U.S. Pat. No. 5,141,718 use more of the well space for the membrane because of an alternate access path to the lower part of the well. However, the membrane cannot occupy the entire cross sectional area of the well because the tubular inserts have walls which themselves occupy well space.
Another problem with inserts involves the means for accessing the lower chambers of wells. Inserts allow access to the lower portion of a well through openings in downwardly extending walls of the insert. While accessing the lower chamber, an unsteady hand may cause a pipette or syringe needle to hit the side of the opening and thus jar the entire insert, which may disturb the membrane substrate and cause damage to the cell later itself. Insert strips allow access to the lower chamber through an extension of a reservoir into which the strips are inserted. However, as with the well inserts, a pipette or syringe may disturb the membrane attached to the insert strip because access to the reservoir is immediately adjacent the insert.
The present invention solves both of these problems by supplying a plate with a membrane that covers the entire cross sectional area of a well of a standard 96-well format, while allowing access to the lower part of the well through distinctly separate access ports. The invention combines the industrial assay advantages of the standardized 96-well cluster plate with the functional advantages of cell culture inserts and insert strips.